The thymic epithelial cells (TECs) are responsible for regulating the development of immature thymocytes into functional self-tolerant mature T cells. A common endoderm-derived TEC progenitor was recently shown to give rise to both cortical and medullary TEC subsets, however, the signaling pathways and cell-to-cell interactions that regulate this process are poorly defined. TEC development and organization is dependent on crosstalk signals received from developing thymocytes as they migrate through the epithelial network. The goal of this study will be to define both the nature and function of the SP thymocyte/TEC interactions that regulate development of the thymic medulla, the thymic microenvironment that is critically responsible for central tolerance. The medullary reconstitution induced by adoptive transfer of mature T cells into mice with blocks in T cell development will be used to test the contribution of specific T cell/TEC interactions. We propose to: (1) use MHC class I / MHC class II double KO mice to determine if the expansion and differentiation of the thymic medulla is dependent on TCR/MHC interactions and identify the specific contributions of CD4 SP, CDS SP and Tregs, in this process;(2) use both fetal thymic organ cultures and medullary reconstitution assays in a newly developed Wnt signaling reporter mouse strain, to access the contribution of Wnt signaling in mTEC development and determine if soluble Wnt inhibitory molecules secreted by SP thymocytes inhibit the canonical Wnt signaling pathway in TECs, thereby providing a molecular switch that is necessary for mTEC development from precursor cells. Fully understanding the signaling pathways and cellular interactions which contribute to TEC development and organization will be critical to designing rational therapeutic strategies to counteract age-associated thymic involution and thymic architecture defects associated with autoimmunity. These strategies will also significantly impact the effectiveness of bone marrow transplantation for cancer treatment by counteracting the severe premature thymic degeneration associated with preparative cytoablative treatments including chemotherapy and radiation, as well as post-transplant GVHD.